The present invention relates to recombinant mycobacterial strains overexpressing essential biosynthetic enzymes of pathogenic mycobacteria and to methods for using these strains. More specifically, the present invention relates to recombinant mycobacteria strains overexpressing D-alanine ligase (Ddl) and the use of such strains in in vitro methods for identifying antimycobacterial agents directed against Ddl.
The bacterial cell wall is an ideal target for drug design since similar structures and biosynthetic pathways are absent from mammalian hosts. The lipid-rich mycobacterial cell wall acts as an efficient permeability barrier (Brennan and Nikaido, 1995). Peptidoglycan, the backbone of this structure, contains the D-amino acids D-alanine, D-glutamate, and diaminopimelate, which may contribute to its stability against proteolytic degradation. D-Alanine is one of the central molecules of the cross-linking step of peptidoglycan assembly. Peptidoglycan biosynthesis in mycobacteria follows pathways similar to those in other eubacteria (Belanger and Inamine, 2000). There are three enzymes involved in the D-alanine branch of peptidoglycan biosynthesis: the pyridoxal phosphate-dependent D-alanine racemase (Alr), the ATP-dependent D-alanine:D-alanine ligase (Ddl), and the ATP-dependent D-alanine:D-alanine-adding enzyme (MurF) (Walsh, 1989). D-Cycloserine (DCS; 4-amino-3-isoxazolidinone) is a rigid analog of D-alanine and targets both Alr and Ddl in Escherichia coli (Lambert and Neuhaus, 1992; Neuhaus, 1967). DCS also inhibits Mycobacterium tuberculosis Alr and Ddl enzymes (David et al., 1969; Strych et al., 2001), suggesting that both Alr and Ddl are targets of DCS in mycobacteria.
DCS is effective against mycobacteria and is recommended to treat multidrug-resistant M. tuberculosis in the DOTS-Plus management plan (Farmer, 2001; World Health Organization, 2000). However, undesirable side effects restrict its use in human chemotherapy (Yew et al., 1993). Nonetheless, the potent bactericidal effect of DCS against mycobacteria makes this drug an attractive prototype compound to develop novel antimycobacterial agents. In addition, identification of the lethal target(s) of DCS action would allow for the rational design of new antimycobacterial drugs, structurally related or unrelated to DCS, targeting enzymes of the D-alanine pathway of peptidoglycan biosynthesis. Moreover, these types of inhibitors may weaken the cell wall and act synergistically with other antimicrobial agents (Rastogi et al., 1990). In the early 1970s, David (1971) isolated and characterized step-wise DCS-resistant M. tuberculosis mutants that showed either normal or reduced cellular permeability to DCS and speculated that Alr plays only a minor role in the mechanism of action of DCS. Mycobacterium smegmatis, a nonpathogenic species, is a useful model to study drug resistance mechanisms in pathogenic mycobacteria, especially when conserved cellular processes are involved (Reyrat and Kahn, 2001; Tyagi and Sharma, 2002). Peteroy et al. (2000) described the isolation and characterization of an M. smegmatis mutant resistant to both DCS and vancomycin though the molecular basis of the resistance mechanism remains unknown. In previous studies, we identified a spontaneous DCS-resistant M. smegmatis mutant with a promoter-up mutation in the alr gene, resulting in the overproduction of the Alr enzyme (Caceres et al., 1997). Alr was shown to be inhibited by DCS in a concentration-dependent manner, and DCS resistance could be conferred to pathogenic mycobacteria carrying the M. smegmatis alr gene in a multicopy plasmid. In addition, DCS was shown to competitively inhibit the native Ddl enzyme from M. tuberculosis (David et al., 1969). Belanger et al. (2000) reported the characterization of a temperature-sensitive M. smegmatis mutant with a single amino acid substitution in Ddl. The mutant was more susceptible to DCS, and the temperature sensitivity phenotype was due to the decreased activity of the mutated enzyme.
Recently, we observed that M. smegmatis alr null mutants are not dependent on D-alanine for growth, suggesting the existence of another pathway for D-alanine biosynthesis (Chacon et al., 2002). In addition, the alr null mutant is hypersusceptible to DCS, suggesting that a lethal target other than Alr is responsible for the bactericidal effect of DCS.
In order to develop novel antimicrobial agents structurally related to DCS, the lethal target(s) of DCS need to be identified and methods of screening for inhibition need to be developed.